Background

Adipose tissue normally contains immune cells that regulate adipocyte function and contribute to metabolic disorders including obesity and diabetes mellitus. Psoriasis is associated with increased risk for metabolic disease, which may in part be due to adipose dysfunction, which has not been investigated in psoriasis. There is currently no standardized method for immunophenotyping human adipose tissue. In prior studies, characteristic phenotypic markers of immune cell populations identified in animal models or in other human tissues have been applied in a similar manner to human adipose tissue. Rarely have these populations been verified with confirmatory methodologies or functional studies. Thus, we performed a comprehensive phenotypic and functional analysis of immune cell populations in psoriatic adipose tissue.

Results

These analyses revealed a wide range of cell surface receptors on adipose tissue macrophages, which may serve a dual purpose in immunity and metabolism. Further, both CD16+CD56Lo and CD16-CD56Hi NK cells were found to correlate inversely with body mass index. The relationship between the predominant CD16+CD56Lo NK cell population and body mass index persisted after adjusting for age, sex, diabetes, and tobacco use.

Conclusions

Together, these studies enhance our understanding of adipose immune cell phenotype and function, and demonstrate that examination of adipose tissue may provide greater insight into cardiometabolic pathophysiology in psoriasis.

Immunophenotyping studies of human adipose have generally assumed that characteristic markers of immune cells described in animal models or in other human tissues can be applied similarly to adipose tissue. A major limitation of prior studies has been a paucity of data confirming flow cytometric analysis with alternative methods of cellular identification. Further, the cadre of markers identified on human adipose immune cells remains limited. Here, we have overcome these deficiencies by utilizing cutting-edge conventional and imaging flow cytometry to characterize the immune cell content, phenotype, and function in adipose specimens from patients with the inflammatory skin condition psoriasis, which is associated with an increased risk of cardiometabolic disease (CMD), [33] and adipose tissue dysfunction [34]. We have identified innate and adaptive immune cell populations, and present a panel of ATM markers that may have dual roles in metabolism and immunity. We have also characterized a NK cell population that correlated inversely with body mass index (BMI). Together, these studies are the first to characterize the immune cell repertoire within psoriatic adipose tissue, and may offer insight into the mediators of CMD in psoriasis.

Study population

Subcutaneous gluteal adipose tissue biopsies were performed in psoriasis patients (n = 30) aged 18 to 70 years in a consecutive sample from the Psoriasis Atherosclerosis and Cardiometabolic Disease Initiative (PACI; NCT01778569). A dermatologist confirmed the diagnosis of plaque psoriasis, and performed body surface area (BSA) and Psoriasis Area and Severity Index (PASI) assessments. Psoriatic arthritis was confirmed by a rheumatologist. Exclusion criteria included history of another systemic inflammatory illness, myocardial infarction, stroke, or chronic infectious disease. Study approval was obtained from the National Heart Lung and Blood Institute (NHLBI) institutional review board in accordance with the Declaration of Helsinki. All study participants provided written informed consent.

Serum factor determination

Adipose tissue immune cell preparation

Adipose tissue specimens were collected in RPMI medium and minced into fine pieces using scissors after careful removal of infiltrating blood vessels with forceps and scissors. Samples were digested in 5 mg/mL Type IV collagenase (Life Technologies, Grand Island, NY) for 30 minutes at 37°C in an Eppendorf thermomixer (Sigma-Aldrich, St. Louis, MO) at 1300 RPM. Tissue fragments were passed through a 40 μM nylon filter (BD Falcon, Beford, MA), washed twice with 1X PBS, and the floating adipocyte fraction was removed by vacuum aspiration. Blood contamination was determined by the visible presence of large amounts of erythrocytes in the cell pellet after digestion and centrifugation. Further, blood contaminated samples demonstrated abundant granulocyte populations by flow cytometry that are not typically present in adipose tissue. Three adipose samples were excluded from the analyses due to blood contamination.

DMARD therapy denotes methotrexate use, except for 1 patient who was taking both methotrexate and hydroxychloroquine. Biologic therapy indicates active TNF antagonist or anti-IL-12/23 receptor use except for one patient who was treated with abatacept for psoriatic arthritis.

Flow cytometric characterization of macrophages, NK cells, and neutrophils in a representative sample of psoriatic adipose tissue. After exclusion of debris, doublets, and non-viable cells, cells were identified as follows: neutrophils = CD14-CD15+CD16+SSCHigranzyme B+, NK cells = CD14-CD15-CD16+/−CD56+/−SSClogranzyme B+, adipose tissue macrophages (ATM) = CD14+CD15-CD16-, which were further gated into 3 subpopulations based on HLADRII and CD206 expression. IL-1β and IL-8 intracellular staining is presented for the total ATM population. All cell populations are presented as percentages of viable cells except for IL-1β and/or IL-8 expressing ATM, and Granzyme B expressing neutrophils, which are reported as percentages of the parent population. Positive gating for each fluorochrome parameter was established using individual fluorescence minus one (FMO) controls.

Confirmation of immune cell populations in psoriatic adipose tissue using imaging flow cytometry. Morphologic and staining characteristics of various cell populations delineated by conventional flow cytometry (Figures 1 and 2) were confirmed in psoriatic adipose tissue using imaging flow cytometry. Cells were gated for positive nuclear staining that did not saturate the camera. Out-of-focus cells were excluded using the gradient RMS function of the brightfield microscopy field. Doublets and debris were eliminated by plotting cell brightfield area against aspect ratio. To determine nuclear morphology population characteristics, manually selected mononuclear or polymorphonuclear cells were identified and hand tagged populations were created. The Feature Finder wizard in IDEAS software was then used to distinguish cells with similar morphologic characteristics in the source population using the circularity and bright detail intensity features on the Hoechst imagery. Three populations of cells were identified: CD3+CD16- T cells (panel D), CD3-CD16+ cells, and CD3-CD16- cells. CD3-CD16- cells were then gated on CD14+ cells (macrophages), followed by sub-gating based on CD206 and HLADRII (DRII) staining (panels A-C). Positive gating for each fluorochrome parameter was established using FMO controls. Percentages of cells in each gate are presented as percentages of nucleated, focused, single cells.

Nuclear morphologic analysis of CD16+ cell populations in adipose tissue confirms flow cytometric phenotypic analysis. Imaging flow cytometry of adipose tissue immune cells was performed as in Figure 3. After exclusion of non-nucleated cells and Hoechst saturating the camera, poorly focused cells, and debris/doublets, CD3-CD14- cells were gated on CD16+ cells. CD16+ cells (~30 cells for each group) were manually selected (Tagged) based on their nuclear morphology to distinguish mononuclear (Circular, panel A) from polymorphonuclear (Polymorph, panel B) cells. The Feature Finder wizard in IDEAS software was used to identify similar cells in the total CD16+ source population (Automated, panels C, E) and to exclude debris and unfocused cells (Automated, panel D). Circularity and Bright Detail Intensity of the Hoechst imagery were the 2 characteristics that best distinguished the manually selected Polymorph and Circular cells. A plot of these 2 parameters for the Tagged and Automated CD16+ populations is depicted. BF = brightfield microscopy, Hoec = Hoechst nuclear staining. CD16 was plotted against SSC to compare Circular and Polymorph populations. Positive gating for each fluorochrome parameter was established using FMO controls.

Comparison of adipose immune cells in psoriasis and controls

To determine whether adipose immune cell composition is affected by psoriasis, we performed a nested case°Control study (n = 6 non-diabetic control and psoriasis patients) matched for age, sex, BMI, smoking, and dyslipidemia. We observed that CD16-CD56Hi NK cells, but not other immune cells, were statistically greater (0.42% live cells in psoriasis versus 0.06% live cells in controls, p = 0.014) in psoriatic compared to control adipose. While the frequencies of total ATM were similar in psoriasis and controls (10.2% live cells in psoriasis versus 12.9% live cells in controls versus, p = 1.0), IL-1β producing (46.1% of psoriatic ATM versus 29.3% of control ATM, p = 0.076) and IL-8 producing (66.1% of psoriatic ATM versus 51.0% of control ATM, p = 0.175) ATM were more abundant in psoriatic compared to control adipose tissue. However, only the HLADRII+CD206+ ATM subset demonstrated a statistically significant difference in IL-1β (40.3% in psoriasis versus 19.6% in controls, p = 0.047), but not IL-8 (62.5% in psoriasis versus 45.7% in controls, p = 0.117), production between psoriatic and control adipose tissue.

We utilized cutting-edge imaging technologies to demonstrate that psoriatic adipose tissue contains immune cells that may influence CMD in psoriasis. Using this approach, we have: (1) identified previously unappreciated immune cell populations and cell surface receptors (2) utilized a combination of phenotypic markers and functional studies to validate those populations (3) described an inverse relationship between psoriatic adipose CD16+CD56Lo NK cells and obesity. We believe these studies take a critical first step toward understanding CMD in psoriasis, and may lead to novel therapies targeting these disorders.

Here, we distinguished 3 populations of ATM expressing varying levels of multi-functional receptors (CD36, LOX-1, MSR1, RAGE, and TLR2) that may perform dual roles in immunity and metabolism. For example, the scavenger receptors MSR1 [37], CD36 [38], and LOX-1 [39] have been shown to bind modified LDL particles, which play a prominent role in CMD and coronary artery disease [40]-[43]. Furthermore, mRNA levels of MSR1, CD36, and LOX-1 in whole adipose tissue specimens have been associated with obesity and insulin resistance in humans [44]. These pleiotropic scavenger receptors also demonstrate important functions in innate immune defense [45]. Similarly, RAGE [46],[47] and TLR2 [48],[49] have essential roles in immunity and CMD. Thus, our data support a growing body of literature indicating that macrophages may directly modulate adipocyte function [50]. Our findings also suggest that ATM populations may represent unique functional subsets that respond to different stimuli, and extend well beyond the oversimplified M1/M2 macrophage paradigm [51]-[53]. This notion could be addressed by exploring global gene expression patterns from purified ATM populations both in the basal state and after activation with various ligands. Importantly, we also demonstrated that psoriatic ATM may be predisposed toward pro-inflammatory cytokine expression, which could contribute to adipose dysfunction in this disorder.

Several other populations of immune cells were identified using conventional and imaging flow cytometry. We are the first to quantify frequencies of FoxP3+ Tregs [previously reported by FoxP3 mRNA expression [29],[30]] and γδ T cells in human adipose tissue. It is also noteworthy that CD16+ cells were primarily neutrophils and NK cells. Bourlier et al. previously found that up to 25% of ATM express CD16 [51]. However, control staining for CD16 was not presented in their manuscript. In our experience, staining with anti-CD16 antibodies in adipose tissue can give high background signal and thus requires careful antibody titration, Fc Receptor saturation, and appropriate control stains. Both conventional and imaging flow cytometric analyses confirmed that CD16 expression on ATM is rare. Low frequencies of B cells [25], NKT cells [21], and neutrophils [26],[27] were also apparent in psoriatic adipose tissue, confirming prior studies in humans. Adipose CD3+ T cells were predominantly αβ T cells and were largely either effector or memory in phenotype, as previously reported [25]. Consistent with previous reports [3],[25], CD4+ T cells were well represented followed in descending order of frequency by CD8+ T cells, Tregs, and γδ T cells. The nature of the cognate antigens driving T cell priming locally and/or prior to migration into adipose tissue remains to be determined.

Adipose NK cells may contribute to obesity in humans. Two populations of NK cells have previously been identified, and are designated as CD16-CD56Hi[25],[31],[32] and CD16+CD56Lo[31] cells. We demonstrated that the predominant CD16+CD56Lo NK cell population correlated inversely with BMI in both unadjusted analyses and after adjustment for CMD risk factors and psoriasis treatment. These findings corroborate those of O’Rourke et al., who demonstrated that the CD16+CD56Lo subset was reduced in obese compared to lean adipose tissue [31]. In contrast, these investigators found increased percentages of CD16-CD56Hi cells in adipose tissue from obese compared to lean patients [31], while Duffaut and colleagues reported no differences in CD56+ NK cells (CD16 expression was not analyzed) irrespective of BMI [25]. Despite these inconsistencies, there is an inverse relationship between adiposity and the major population of adipose NK cells (CD16+CD56Lo). Future studies in animal models should directly test whether NK cell deficiency impacts adipose tissue composition and function. Two indirect lines of evidence have suggested this to be the case. First, studies of the NK cell growth factor IL-15 have shown that IL-15 overexpression increased adipose NK cell infiltration and decreased adipose tissue mass in mice [54], although the latter outcome may have been due to a direct effect of IL-15 on adipocytes [55]. Second, leptin receptor mutant animals demonstrated lower circulating and tissue NK cell numbers and impaired NK cell function as well as prominent obesity and insulin resistance [56]. Together, these studies suggest that NK cells may protect against obesity in mouse and man.

We acknowledge that this study had certain limitations such as modest sample size and single-center study design, which could affect the generalizability of the results. However, we provide unique data in 30 psoriatic patients under standardized study conditions (NCT01778569). Another consideration is that therapies for psoriasis and/or CMD could impact adipose tissue cell populations. Our study used subcutaneous adipose tissue from psoriasis patients. While visceral adipose tissue is generally considered to be more inflamed, recent studies have revealed that subcutaneous and visceral adipose display a similar pro-inflammatory phenotype, suggesting that experiments using the readily accessible subcutaneous depot will enhance our understanding of adipose biology [57],[58]. Despite these limitations, our data are methodologically sound and may be informative for understanding CMD in psoriasis.

SR is an American Board of Internal Medicine certified rheumatologist, member of the Group for the Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA), and a Metzger Clinical Scholar at the National Institutes of Health. SR has extensive expertise in the clinical, basic, and translational mechanisms linking inflammatory disorders to cardiovascular and metabolic disease.

ES is an Intramural Research Training Awardee (IRTA) at the National Institutes of Health who specializes in flow cytometry.

PKD is a postdoctoral fellow at the National Institutes of Health with significant expertise in flow cytometry, cell biology, and Th17 mediated inflammatory diseases.

LS is a biologist in the National Heart Lung and Blood Institute Flow Cytometry Core who is an expert in imaging flow cytometry.

EW is an Intramural Research Training Awardee (IRTA) at the National Institutes of Health who specializes in adipose tissue procurement and biology.

AJ is a special volunteer at the National Heart Lung and Blood Institute who specializes in adipose tissue procurement and biology.

JD is a biologist at the National Heart Lung and Blood Institute with significant expertise in tissue processing and characterization and flow cytometry.

HBN is an American Board of Dermatology certified dermatologist with extensive expertise in psoriasis, graft-versus host disease, and the neutrophilic dermatoses. HBN is a senior clinical scholar at the National Cancer Institute.

MPP is a staff scientist at the National Heart Lung and Blood Institute who specializes in cellular and molecular biology, lipid biology, vascular biology, and Th17 mediated immune diseases.

JPM heads the National Heart Lung and Blood Institute Flow Cytometry Core and is a key member of the Center for Human Immunology at the National Institutes of Health. JPM is board certified in hematology by the American Board of Bioanalysis and is licensed to direct clinical flow cytometry laboratories in the states of New York and New Jersey. He has edited three books on flow cytometry, is associate editor of Cytometry Part B: Clinical Cytometry, and is past President of the Clinical Cytometry Society. JPM is also a member of numerous professional societies including AACR, ASH, CCS and ASCP, and a recipient of the 2006 Lifetime Achievement Award from the American Society of Clinical Pathology.

NNM is an American Board of Internal Medicine certified cardiologist who specializes in preventative cardiology and cardiovascular imaging. He is the inaugural Lasker Clinical Scholar and section chief in the Section of Inflammation and Cardiometabolic Diseases at the National Heart, Lung, and Blood Institute, National Institutes of Health. He is also a member of Group for the Assessment of Psoriasis and Psoriatic Arthritis (GRAPPA) and the founder of the Psoriasis, Atherosclerosis, and Cardiometabolic Disease Initiative (NCT01778569).

Acknowledgements

This work was funded using intramural funds from the National Heart Lung and Blood Institute (NHLBI DIR grant HL006193-01), the National Institute of Arthritis and Skin and Musculoskeletal Diseases, and the National Cancer Institute.

12967_2014_258_MOESM4_ESM.tiffAdditional file 4: Figure S1.: The majority of Tregs in psoriatic adipose tissue are CD4+. Multi-parameter flow cytometry was performed as in Figure 2. After sequentially gating out debris, doublets, and non-viable cells, Treg cells were identified as CD3+CD16-CD19-CD56-TCRγδ-FoxP3+ cells. CD4+ and CD8+ Tregs from a representative sample are depicted with cell frequencies presented as percentages of the parent population. Positive gating for each fluorochrome parameter was established using FMO controls. (TIFF 4 MB)

12967_2014_258_MOESM5_ESM.tiffAdditional file 5: Figure S2.: PD-1 is highly expressed on adipose tissue B cells. Multi-parameter flow cytometry was performed as in Figure 2. After sequentially gating out debris, doublets, and non-viable cells, B cells were identified as CD3-CD16-CD19+CD56- cells with high surface expression of the B cell marker PD-1. A representative sample is depicted, with cell frequencies presented as percentages of the parent population. Positive gating for each fluorochrome parameter was established using FMO controls. (TIFF 3 MB)

12967_2014_258_MOESM6_ESM.tiffAdditional file 6: Figure S3.: Specificity of adipose tissue macrophage markers. Imaging flow cytometric analysis of psoriatic adipose tissue was performed to determine the morphologic and staining characteristics of CD16+ cells delineated by flow cytometry (Figures 1 and 2). After exclusion of non-nucleated cells and Hoechst saturating the camera, poorly focused cells, and debris/doublets, CD3-CD14- cells were sub-gated based on HLADRII (DRII) and CD206 staining. CD3-CD14+ cells were also examined for CD16 expression. Cell frequencies are presented as percentages of nucleated, focused, single cells. Positive gating for each fluorochrome parameter was established using FMO controls. (TIFF 3 MB)

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